Mammalian cell membranes perform very important functions relating to the structural integrity and activity of various cells and tissues. Of particular interest in membrane physiology is the study of trans-membrane ion channels which act to directly control a variety of physiological, pharmacological and cellular processes. Numerous ion channels have been identified including calcium (Ca), sodium (Na) and potassium (K) channels, each of which have been analyzed in detail to determine their roles in physiological processes in vertebrate and insect cells.
A great deal of attention has recently been focused on the potassium channel because of its involvement in maintaining normal cellular homeostasis. A number of these potassium channels open in response to changes in the cell membrane potential. Many voltage-gated potassium channels have been identified and are distinguishable based on their electrophysiological and pharmacological properties. An extended family of at least twenty genes have been isolated, each encoding functionally distinct voltage-gated potassium channels, and each with a unique tissue distribution pattern. Several of these have been shown to be involved in maintaining the cell membrane potential and controlling the repolarization of the action potential in neurons, muscle and pancreatic .beta.-cells. Potassium currents have been shown to be more diverse than sodium or calcium currents and also play a role in determining the way a cell responds to an external stimulus. The diversity of potassium channels and their important physiological role highlights their potential as targets for developing therapeutic agents for various diseases.
Type II or non-insulin-dependent diabetes (NIDDM) is a chronic and debilitating disorder affecting at least 5% of the human population (Bell, G. I. et al., 1980, Nature 284:26 and Horst-Sikorska, W. et al., 1994, Hum. Genet. 93:325). NIDDM, manifested as fasting hyperglycemia, results either from a defect in insulin release from pancreatic .beta.-cells or from the inability of peripheral tissues to respond appropriately to insulin (Bell, G. I. et al., 1980, supra, Horst-Sikorska, W. et al., 1994, supra and Herman, W. H. et al., 1994, Diabetes 43:40).
Current therapeutic management of this disease is based primarily on the use of drugs (sulfonylurea compounds) that enhance insulin release by selectively modulating K.sub.ATP channels (Boyd III, A. E., 1988, Diabetes 37:847, Rajan, A. S. et al., 1990, Diabetes Care 13:340, Misler, S. et al., 1986, Proc. Natl. Acad. Sci USA 83:7119, Petersen, O. H. and Findlay, I., 1987, Physiol. Rev. 67:1054 and Ashcroft, F. M., 1988, Ann. Rev. Neurosci. 11:97). Hypoglycemia is a frequent side effect of such anti-diabetic therapy because these drugs, mimicking the action of glucose, induce membrane depolarization of .beta.-cells (Bell, G. I. et al., 1980, supra, Horst-Sikorska, W. et al., 1994, supra and Herman, W. H. et al., 1994, supra, Boyd III, A. E., 1988, supra, Rajan, A. S. et al., 1990, supra, Misler, S. et al., 1986, supra, Petersen, O. H. and Findlay, I., 1987, supra, Ashcroft, F. M., 1988, supra, Dukes, I. et al., 1994, J. Biol. Chem. 269:10979, Cook, D. L. et al., 1991, Trends Neurosci. 14:411, Smith, P. A. et al., 1990, J. Gen. Physiol. 95:1041, Smith, P. A. et al., 1990, FEBS Lett. 261:187, Atwater, I. et al., 1983, Cell Calcium 4:451, Ammala, C. et al., 1991, Nature 353:849 and Worley III, J. F. et al., 1994, J. Biol. Chem. 269:12359). Sulfonylurea-induced insulin release, therefore, occurs in a glucose-independent manner. A glucose-dependent insulin secretagogue could potentially avoid the debilitating side effect of hypoglycemia, and would therefore be extremely useful.
Another form of treatment in severe long-standing NIDDM is insulin replacement. This approach, although effective, is time-consuming, expensive and requires the administration of painful injections often many times daily. To say the least, NIDDM patients would welcome a more effective treatment with fewer side effects. An understanding of the mechanisms responsible for insulin secretion may help identify new targets for the development of such novel anti-diabetic drugs.
Transmembrane ion channels are the primary elements that transduce signals in pancreatic .beta.-cells, resulting in the release of insulin (Boyd III, A. E., 1988, supra, Rajan, A. S. et al., 1990, supra, Misler, S. et al., 1986, supra, Petersen, O. H. and Findlay, I., 1987, supra , Ashcroft, F. M., 1988, supra, Dukes, I. et al., 1994, supra, Cook, D. L. et al., 1991, supra, Smith, P. A. et al., 1990, J. Gen. Physiol. 95:1041, Smith, P. A. et al., 1990, FEBS Lett. 261:187, Atwater, I. et al., 1983, supra, Ammala, C. et al., 1991, supra and Worley III, J. F. et al., 1994, supra.). In response to an elevation in external glucose, the .beta.-cell membrane slowly depolarizes (phase I). This metabolic coupling appears to be due to an increase in cytosolic ATP, which results in the closure of ATP-sensitive potassium (K.sub.ATP) channels. The membrane depolarization in turn initiates sinusoidal bursts of calcium action potentials (phase II), during which intracellular calcium rises, triggering insulin secretion (Boyd III, A. E., 1988, supra, Rajan, A. S. et al., 1990, supra, Misler, S. et al., 1986, supra, Petersen, O. H. and Findlay, I., 1987, supra, Ashcroft, F. M., 1988, supra, Dukes, I. et al., 1994, supra, Cook, D. L. et al., 1991, supra, Smith, P. A. et al., 1990, J. Gen. Physiol. 95:1041, Smith, P. A. et al., 1990, FEBS Lett. 261:187, Atwater, I. et al., 1983, supra, Ammala, C. et al., 1991, supra and Worley III, J. F. et al., 1994, supra). Voltage-gated potassium channels have been suggested to play a critical role in repolarizing the membrane after each of these calcium spikes.
Alteration in any of these ionic signalling events could interfere with insulin release and result in hyperglycemia. Overexpression of voltage-gated potassium channels, for example, might be expected to excessively hyperpolarize the membrane following each calcium spike and thereby inhibit the reopening of voltage-gated calcium channels with the reduction in calcium entry leading to diminished insulin release and hyperglycemia. We have therefore focused our attention on identifying the pancreatic islet cell voltage-gated potassium channel.